ENZYMES AS BIOCATALYSTS

Anamika Chatterjee
12BCH002

Guided by:

Prof. Priya Saxena

WHAT ARE ENZYMES ?

Enzymes are biological molecules that act as

catalysts . Every biochemical reaction in the cell is
catalysed by specic enzymes.
Enzymes are in generalglobular proteinsand range
from just 62 amino acid residues in size,to over
2,500 residues.
Like all proteins, enzymes are long, linear chains of
amino acids thatfoldto produce athree-dimensional
product. Each unique amino acid sequence produces
a specic structure, which has unique properties.

ENZYMES V/S CHEMICAL CATALYSTS

generally more efcient (lower concentration of

enzyme needed)
can be modied to increase selectivity, stability,
and activity
highly selective
environment friendly and
degraded in the environment

are

completely

The most important advantage of a biocatalyst is

its high selectivity.
Such high selectivity is very desirable in
chemical synthesis as it may offer several benets
such as reduced or no use of protecting groups,
minimized side reactions, easier separation, and
fewer environmental problems.
Other advantages, like high catalytic efficiency
and mild operational conditions, are also very
attractive in commercial applications.

LOCK & KEY MODEL

suggested byEmil Fischerin 1894

both the enzyme and the substrate possess
specic complementary geometric shapes that t
exactly into one another
while this model explains enzyme specicity, it
fails to explain the stabilization of the transition
state that enzymes achieve

INDUCED FIT THEORY

Daniel Koshlandsuggested a modication to the

lock and key model: since enzymes are rather
flexible structures, the active site is continuously
reshaped by interactions with the substrate as
the substrate interacts with the enzyme.
As a result, the substrate does not simply bind to
a rigid active site; the amino acidsidechainsthat make up the active site are molded
into the precise positions that enable the enzyme
to perform its catalytic function

ENZYME KINETICS
MICHAELIS MENTEN EQUATION

Free enzyme (E),

Substrate (S),
Enzymesubstrate complex (ES) and products (P)

v = k3 [ES]

v max = k 3 ([E] + [ES])

This function represents a typical saturation curve

where Km equates to the substrate concentration
when the velocity is half the maximum and serves
to indicate the rate at which maximum velocity is
reached (i.e. high Km means a slow rate and vice
versa).

Experimentally determining Rate parameters

Lineweaver Burk plot

gives good estimates on Vm, but not necessarily on

Km

EADIE HOSTFEE PLOT

Eadie Hostfee plots can be subject to large errors since both

coordinates contain but there is less bias on points at low [S].

HANES WOOLF PLOT

This plot is used as the best possible approximation of Vm .

APPLICATIONS

Range of potential applications

Advent of recombinant DNA technology has made
possible to make formerly rare enzymes in large
quantities and hence reduce cost.

Smaller quantities of ne high-purity enzyme

preparations are also required for numerous
applications.
These include roles as therapeutic agents, many of
which are now recombinant products, components
of food and medical diagnostic test kits, biosensors,
as research tools and many other purposes.
There is an increasing demand for ne enzymes
used in molecular biology, particularly restriction
endonucleases and DNA polymerases,

IMMOBILIZATION

Enzymes that are more expensive to produce,

including many intracellular microbial enzymes, and
those employed in biosensors and analytical test
systems, are often used in an immobilized form.

Benecial features:
reuse;
suitability
for application within continuous
operations;
their product is enzyme free, therefore further
processing to remove or inactivate the enzyme is not
required;
improved enzyme stability; and
reduced effluent disposal problems.

ENZYMATIC FUEL CELLS

Advantages :
Enzymes are easily mass manufactured and cheaper
compared to the mining of precious metals which have
an inelastic supply.
Enzymes can also process organic compounds such as
sugars and alcohols, which are exceedingly frequent in
nature.
Most organic compounds cannot be used as fuel with
metal catalysts because the carbon monoxide formed
during the fuel cells working will quickly poison the
precious metals that the cell relies on, rendering it
ineffective.
Sugars and other biofuels can be harvested on a
massive scale, and can be found in nearly any part of
the world, thus making it an amazingly striking option
from a logistics standpoint, and even more so for those
concerned with the adoption ofrenewable energy

Biomass is extensively used as fuels such as in

production of bio- ethanol. However food security
concerns prevent the large scale production of bio
ethanol using sugars and starch. Hence,
lignocellulose biomass is a possible alternative.
Until recently, lignocellulose biomass was inert to
enzymatic hydrolysis. However a patented method
has been developed to produce biothanol from
lignocelluloses biomass. The cellulose is separated
from lignin and further processed to get sugars and
nally ethanol. [8]
Another patent has been extended to use sweet
variety of sorghum for production of biofuels by
increasing the sugar content and decrease
lignocellulose content. [9]

CASE STUDY
Two plant based enzymes with diverse
applications were studied and an economic
analysis was performed to evince that enzymes
are cheaper to produce since the advent of
recombinant technologies and hence a more
viable option over industrial catalysts.

BUTYRYL CHOLINESTERASE
It is an enzyme that can act as a bio scavenger to
offset the effects of cholinesterase inhibitors such
as sarin . Also, it is already in use as bio defense
countermeasures in several countries. At present,
BuChE is extracted from outdated human blood
supplies, but it can also be made recombinantly
in cell culture, transgenic animals, and plant
systems

SARIN

Sarin, is used as achemical weaponowing to its extreme

potency as anerve agentand it has been classied as
aweapon of mass destruction inUN Resolution 687.
Production and stockpiling of sarin was outlawed as of
April 1997 by theChemical Weapons Conventionof 1993,
and it is classied as aSchedule 1 substance.
Sarin can be lethal even at very low concentrations, with
death following within 1 to 10 minutes after direct
inhalation due to suffocation from lung muscle
paralysis .People who absorb a non-lethal dose, but do not
receive immediate medical treatment, may suffer
permanent neurological damage.

With the recent use of chemical nerve agents

such as sarin, there is continued interest on the
part of many governments in stockpiling BuChE
as a countermeasure.
The advantages of speed of prototyping,
manufacturing flexibility, and ease of indoor
scale-up are clearly differentiating features of
transient systems and explain why this approach
has been widely adopted in the manufacture of
many PMP products.

CELLULASES

Cellulase complex, a mixture of 46 enzymes

used to saccharify cellulosic feedstocks for the
manufacture of ethanol as a bio fuel.
This target was chosen for study because, for
more than 30 years, the price of cellulases has
been a major hindrance to the economic
feasibility of cellulosic ethanol programs.
Cellulases provide a good example of a
tremendously cost sensitive product class and
hence serve as a novel enzyme to perform
studies.

Cellulases currently under evaluation in bioethanol programs are

all produced by microbial fermentation.

Despite decades of research on lowering cellulase manufacturing

costs, these enzymes still account for 2040% of cellulosic ethanol
production costs.

Hence, lowering the cost of the biocatalyst is critical to the

eventual adoption of bio fuel processes that utilize renewable
plant biomass feedstocks without competing with food or feed
supplies. An alternative to fermentation produced cellulases is
the production of these enzymes in crop plants, with the ultimate
goal of producing cellulases at commodity agricultural prices.

METHODS
SuperPro Designer ,Version 9.0 (Intelligen, Inc.,
Scotch Plains, NJ; http://www. intelligen.com/),
was the software used for the modelling of both
case studies.
It is generally used for techno economic modeling
and is utilized for process simulation and
flowsheet development. It is capable of
performing
mass
and
energy
balances,
equipment
sizing,
batch
scheduling/debottlenecking, capital investment
and operating cost analysis, and protability
analysis.

Nicotina host plants were the source for obtaining the

active ingredient of both the enzyme classes . Nicotiana
species,

notably

N.

tabacum,

N.

excelciana,

and

N.benthamiana, are preferred hosts for PMB manufacture

due to their metabolic versatility, permissiveness to the
propagation

of

various

viral

replicons,

and

high

expression yields achievable with a wide range of targets.

Use of these hosts for production of clinical trial

materials is also familiar to FDA and other regulatory
agencies, thus facilitating Nicotianas acceptance in
regulation-compliant manufacturing.

Plants are also free of adventitious agents that

can infect humans and animals (a concern in cellbased systems and transgenic animals) and this
inherent safety feature pays dividends by
enabling the streamlined purication of the nal
product without the need for adventitious agent
removal steps.

Plants eukaryotic protein processing enable

them to synthesize complex classes of
biomolecules, such as monoclonal antibodies and
therapeutic enzymes.

Recent advances in glycoengineering of host plants

have enabled the production of human- and
mammalian-identical (or at least mammalian similar)
molecules that exhibit comparable or even superior
pharmacology
to
their
cell
culture-derived
counterparts.
Inescapably, the growth of the population in
developing world regions, the aging of the population
in industrialized countries, population displacement
due to political turmoil, degradation of environmental
quality, and depletion of non renewable resources are
serious challenges that have not been and likely
cannot be readily met only by the existing product
manufacturing
platforms.
This
creates
new
opportunities for plant-based systems to yield lower
cost and more widely accessible biopharmaceuticals,
food, feed, fuels, and industrial materials.

RESULTS

BuChE could be manufactured in plants using

transient expression for approximately $234 per
400-mg dose if an existing toll-manufacturing
facility were available to accommodate production of
25kg/year of puried enzyme (equivalent to 62,500
doses/yr).
If a new facility with that capacity needs to be built,
the cost per dose is projected to increase to
approximately $474.
Further economic gains could be possible if capacity
were to be increased to 100 kg of enzyme per year or
more, which, in a toll manufacturing scenario, could
reduce the cost of BuChE to below $200/dose.

Even with conservative assumptions, these costs

are dramatically below the costs obtainable with
blood extraction processes for this enzyme and
may be substantially lower than those for
transgenic approaches.
In addition, the combination of speed of product
prototyping enabled by transient expression, the
superior quality and functionality of the BuChE
obtained, lack of adventitious agents, and the
rapid scalability of plant systems should make
plants the preferred platform for the rapid and
cost effective production of this and similar
products.

With the assumptions and process parameters

adopted for the cellulose case study , our results
show that high density eld cultivation of tobacco
induced to synthesize several enzymes of the
cellulase complex could be competitive with
fungal cellulases produced by fermentation for
the saccharication of biomass in the production
of cellulosic ethanol

REFERENCES
[1] Tyler Johannes, Michael R. Simurdiak , Huimin
Zhao, Biocatalysis, Encyclopedia of Chemical
Processing, 2006 , pp 102
[2] http://en.wikipedia.org/wiki/Enzyme, 28/01/15,
20:20
[3] Saha B. et al., Applied Biocatalysis in Specialty
Chemicals and Pharmaceuticals; ACS
Symposium Series; American Chemical Society:
Washington, DC, 2001
[4] Kim Gail Clarke, Bioprocess Engineering,
Woodhead Publishing , 2013

[5] http://www.kyrobio.eu/Anton_Glieder.html, 8/4/2015,

20:34
[6] Michael J. Waites, Industrial Microbiology: An
Introduction, Blackwell Science, 2001
[7] http://en.wikipedia.org/wiki/Enzymatic_biofuel_cell,
8/4/2015, 21:00
[8]K. Ties, Method for producing ethanol by
fermentation from lignocellulosic biomass, 2011
[9] M.Joachim, C.T. Martin, B.Remy, Compositions and
Methods for biofuel crops, 2011
[10] Daniel Tus, Tiffany Tu, and Karen A. McDonald,
Manufacturing Economics of Plant-Made Biologics:
Case Studies in Therapeutic and Industrial Enzymes,
Hindawi Publishing Corporation, BioMed Research
International Volume 2014, Article ID 256135,